Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall

Cells across all kingdoms of life actively partition molecules between discrete cellular compartments. In Gram-positive bacteria, a thick and highly cross-linked peptidoglycan cell wall separates the bacterial membrane from the extracellular space, imposing a barrier that must be crossed by proteins whose functions require that they be exposed on the bacterial cell surface1,2. Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA3, remain associated with the bacterial membrane yet somehow thread through tens of nanometers of dense, cross-linked cell wall to expose their N-terminus on the outer surface4,5. Here, we show that entropy can drive the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We develop a physical model predicting that the entropic constraint imposed by a thin periplasm is sufficient to drive translocation of an intrinsically disordered protein like ActA across a porous barrier similar to the cell wall. Consistent with this scenario, we demonstrate experimentally that translocation depends on both the dimensions of the cell envelope and the length of the disordered protein, and that translocation is reversible. We also show that disordered regions from eukaryotic nuclear pore complex proteins are capable of entropy-driven translocation through Gram-positive cell walls. These observations suggest that entropic forces alone, rather than chaperones or chemical energy, are sufficient to drive translocation of certain Gram-positive surface proteins for exposure on the outer surface of the cell wall.

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Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall

Nature Microbiology ◽

10.1038/s41564-021-00942-8 ◽

2021 ◽

Vol 6 (8) ◽

pp. 1055-1065

Author(s):

David K. Halladin ◽

Fabian E. Ortega ◽

Katharine M. Ng ◽

Matthew J. Footer ◽

Nenad S. Mitić ◽

...

Keyword(s):

Cell Wall ◽

Bacterial Cell ◽

Bacterial Cell Wall ◽

Disordered Proteins ◽

Gram Positive

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Structures of the surface exposed proteins of Gram positive bacteria

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095679 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C432-C432

Author(s):

George Minasov ◽

Salvatore Nocadello ◽

Ekaterina Filippova ◽

Andrei Halavaty ◽

Wayne Anderson

Keyword(s):

Cell Wall ◽

Infectious Diseases ◽

Bacillus Anthracis ◽

Structural Genomics ◽

Drug Targets ◽

Morphological Changes ◽

Target Selection ◽

Surface Proteins ◽

Human Pathogens ◽

Gram Positive

The Center for Structural Genomics for Infectious Diseases (CSGID) applies structural genomics approaches to biomedically important proteins from human pathogens. It also provides the infectious disease community with a high throughput pipeline for structure determination that carries out all steps of the process, from target selection through structure deposition. Target proteins include drug targets, essential enzymes, virulence factors and vaccine candidates. The CSGID has deposited over 680 structures in the Protein Data Bank. The proteins that are exposed on the surface of Gram positive bacterial pathogens (including Staphylococcus aureus, Bacillus anthracis, Listeria monocytogenes, Streptococcus species and Clostridium species) have been one focus area for the CSGID. So far, the structures of more than 55 of these proteins have been determined. The surface proteins are important in the interactions between the pathogen and its host, but many of them are as yet functionally uncharacterized. Among the examples that will be presented is the Bacillus anthracis SpoIID protein. SpoIID is part of a coordinated cell wall degradation machine that is essential for sporulation and the morphological changes involved. It represents a new family of lytic transglycosylases that degrade the glycan strands of the peptidoglycan cell wall. The two active site clefts in the dimeric enzyme include residues from both subunits, suggesting that the dimer is required for activity. This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contracts No. HHSN272200700058C and HHSN272201200026C.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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Telavancin, a Multifunctional Lipoglycopeptide, Disrupts both Cell Wall Synthesis and Cell Membrane Integrity in Methicillin-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.3.1127-1134.2005 ◽

2005 ◽

Vol 49 (3) ◽

pp. 1127-1134 ◽

Cited By ~ 291

Author(s):

Deborah L. Higgins ◽

Ray Chang ◽

Dmitri V. Debabov ◽

Joey Leung ◽

Terry Wu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Membrane Potential ◽

Cell Membrane ◽

Bactericidal Activity ◽

Bacterial Cell ◽

Cell Membrane Potential ◽

Gram Positive ◽

Methicillin Resistant ◽

Bacterial Cell Membrane

ABSTRACTThe emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited late-stage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptideN,N′-diacetyl-l-lysinyl-d-alanyl-d-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistantStaphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K+. The timing of these changes correlated with rapid , concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.

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Covalent Sortase A Inhibitor ML346 Prevents Staphylococcus aureus Infection of Galleria mellonella

RSC Medicinal Chemistry ◽

10.1039/d1md00316j ◽

2021 ◽

Author(s):

Xiang-Na Guan ◽

Tao Zhang ◽

Teng Yang ◽

Ze Dong ◽

Song Yang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Galleria Mellonella ◽

Surface Proteins ◽

Sortase A ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Aureus Infection ◽

Cell Wall Peptidoglycan

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation...

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A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

Infection and Immunity ◽

10.1128/iai.72.5.2710-2722.2004 ◽

2004 ◽

Vol 72 (5) ◽

pp. 2710-2722 ◽

Cited By ~ 152

Author(s):

David Comfort ◽

Robert T. Clubb

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Surface Proteins ◽

Sequence Motifs ◽

Comparative Genome Analysis ◽

Gram Positive ◽

Microbial Genomes ◽

Searchable Database ◽

Gram Positive Bacteria ◽

Related Proteins

ABSTRACT Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only ∼20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/ ) in a searchable database.

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